Structure-based optimization of type III indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors

Abstract The haem enzyme indoleamine 2,3-dioxygenase 1 (IDO1) catalyses the rate-limiting step in the kynurenine pathway of tryptophan metabolism and plays an essential role in immunity, neuronal function, and ageing. Expression of IDO1 in cancer cells results in the suppression of an immune response, and therefore IDO1 inhibitors have been developed for use in anti-cancer immunotherapy. Here, we report an extension of our previously described highly efficient haem-binding 1,2,3-triazole and 1,2,4-triazole inhibitor series, the best compound having both enzymatic and cellular IC50 values of 34 nM. We provide enzymatic inhibition data for almost 100 new compounds and X-ray diffraction data for one compound in complex with IDO1. Structural and computational studies explain the dramatic drop in activity upon extension to pocket B, which has been observed in diverse haem-binding inhibitor scaffolds. Our data provides important insights for future IDO1 inhibitor design.


Introduction
Immuno oncology provides powerful therapies against cancer in the form of immune checkpoint inhibitors, adoptive cell therapies, monoclonal antibodies, oncolytic viruses, cancer vaccines, and other immuno modulators. However, low response rates due to tumoral immune suppression and resistance remain an unsolved issue 1 . L-Trp catabolism along the kynurenine pathway is an important mechanism employed by cancer cells to escape a potentially effective immune response 2,3 . The rate-limiting step in this pathway is catalysed by indoleamine 2,3-dioxygenase 1 (IDO1) and by tryptophan 2,3-dioxygenase (TDO), with the IDO1 paralogue IDO2 also potentially playing a role 4,5 Preclinical data suggests that a combination of IDO1 inhibitors with other anticancer agents results in effective anti-tumor immunity [6][7][8] . However, in a phase 3 clinical trial of the IDO1 inhibitor epacadostat in combination with pembrolizumab in melanoma patients, the combination failed to increase the overall and progressionfree survival when compared to pembrozilumab alone 9 . This failure highlighted the need for a better understanding of the role of the kynurenine pathway, for the development of better IDO1 inhibitors, and for improved trial design 10,11 . Additional aspects of IDO1 biology are constantly discovered and may influence its role in cancer, such as its signalling activity [12][13][14] , regulation by haem availability 15 , nitrite reductase activity in hypoxic tissues 16 , involvement in the redox signalling pathways of hydrogen peroxide and singlet oxygen 17 , and activation by polysulphides 18 . Based on the ongoing interest for IDO1 inhibitors capable of modulating these different pathways and processes selectively, a multitude of smallmolecule IDO1 inhibitors have been described 19,20 , and more than 60 crystal structures of IDO1 have been deposited in the protein data bank (PDB) 21 . These structures with a large diversity of bound cofactors and ligands, including the clinical-stage IDO1 inhibitors epacadostat (1, INCB024360, Figure 1A) 23 , navoximod (2, NLG-919/GDC919) 24 , EOS200271 (3) 25 , and linrodostat (4, BMS-986205) 26 , yield a wealth of information for inhibitor design 27 .
We have previously classified IDO1 inhibitors into four types according to their preferential binding and inhibition mechanism 22 . Type i inhibitors (e.g. 1-methyl-L-tryptophan) preferentially bind to oxygen-bound holoIDO1, type ii inhibitors (e.g. epacadostat) to free ferrous holoIDO1, type iii inhibitors (e.g. navoximod) to free ferric holoIDO1, and type iv inhibitors (e.g. linrodostat) to apoIDO1. Lead optimisation of haem-iron binding type ii and type iii inhibitors has proven difficult due to the selectivity and sensitivity of the haem-ligand interactions to changes in the electronic structure of the ligand 28 and due to the small size of the distal haem pocket (pocket A) 29 , crucial for inhibitor activity. The IDO1 active site further comprises Pocket B, which extends from pocket A towards the entrance of the active site ( Figure 1B,C), and whose size and shape are determined by the conformation of the flexible JK-loop 27 . Although its influence on inhibitor affinity is less pronounced than pocket A, it is of interest for modulation of other compound properties such as specificity, absorption, distribution, metabolism and excretion (ADME), and pharmacokinetics/pharmacodynamics (PK/PD).
Despite the large number of published IDO1 inhibitors, there are only a very limited number of sub-micromolar type ii and type iii inhibitor scaffolds known. Epacadostat (1) binds to the haem iron through an unusual high-affinity hydroxyamidine scaffold, which was discovered by Incyte 23,30 and has later been exploited by other groups [31][32][33][34][35][36][37][38] . Due to its unique tilted iron-binding conformation, it provides a straightforward access to pocket B and allowed the development of numerous nanomolar IDO1 inhibitors with a high selectivity over TDO 27 . Except for the hydroxyamidines, almost all nanomolar haem-binding IDO1 inhibitors are based on a haem-binding free or fused azole scaffold.
Imidazoles are classical haem binders, as present in the haembinding histidine side chain and in many antifungal drugs, and 4phenyl-imidazole 39 was the first cocrystallized IDO1 inhibitor 40 . Early structure-based optimisation of this chemotype yielded the 2-hydroxy-substituted phenyl derivative as the most active  22 ; ligand MMG-0358). (D) Main lead optimisation strategies pursued in this work. The red arrow denotes a preferentially hydrogen-bonding substituent, the green arrow a hydrophobic substituent, and the blue arrows potential access points to pocket B. As demonstrated before, the acidic hydrogens on the triazole rings are crucial for activity and therefore cannot provide access to pocket B. compound 41 . Combination of the 2-hydroxy substitution with a 5-chloro substitution led to the most efficient imidazole compound (5) with a ligand efficiency (LE) of 0.68 kcal/mol/heavy atom (HA) 24,28 . In subsequent developments, extending the scaffold to pocket B, a drop in LE was always encountered 42 , but nanomolar activities could be obtained in some 1,5-disubstituted imidazoles 43,44 . Rigidification of the scaffold to imidazo [5,1-a]isoindole also allowed to retain nanomolar activities [45][46][47][48][49] and led to the development of navoximod (2, Figure 1) 24 . Independently from the work on imidazo [5,1-a]isoindoles, structural and functional data of imidazo [2,1-b] [1,3]thiazole based IDO1 inhibitors was disclosed in 2014 (6) 50 . Subsequent work on this fused imidazole scaffold led for example to compound 7 with a good enzymatic activity and a LE of 0.41 kcal/mol/HA 51 . However, compounds with this scaffold lack cellular activity [51][52][53] . Indazoles are also known haem binders, and the indazol-4amine scaffold developed by IOmet Pharma 54 yielded selective nanomolar TDO inhibitors and dual IDO1/TDO inhibitors [55][56][57] . Xray structures of the complexes between IDO1 and some indazol-4-amines such as compound 8 have recently been resolved 57 . These compounds provide access to pocket B while preserving a LE of up to 0.47 kcal/mol/HA.
We have previously discovered 1,2,3-triazoles as highly efficient IDO1 inhibitors and resolved the X-ray structure of MMG-0358 (9) in complex with IDO1 ( Figure 1) 22,28,29,58 . Compound 9 forms a direct bond to the haem iron, a hydrogen bond with Ser167 through its hydroxy function, and hydrophobic interactions through its chloro substituent, leading to a LE of 0.76 kcal/mol/HA. A few 4,5-disubstituted 1,2,3-triazoles with nanomolar activities have been reported 59 , but as we detail below, we failed to reproduce these results. Interestingly, the N-phenyl-1,2,3-triazol-4-amine (10) developed by Vertex 60 demonstrates an IDO1 inhibition mechanism distinct from the 4-phenyl1,2,3-triazoles despite a similar binding mode 22 . 1,2,4-Triazole is a common haem-binding scaffold present in many antifungal drugs such as fluconazole, and its direct iron binding in sterol 14a-demethylase (CYP51) is well documented by structural data. However, these 1-substituted 1,2,4-triazoles have been found to be inactive on IDO1, at variance with imidazole antifungals such as miconazole, which showed some activity 61 . However, we recently demonstrated that the 3-substituted 1,2,4triazole scaffold can provide highly efficient IDO1 inhibitors. Two simple substitutions on the 3-phenyl-1,2,4-triazole scaffold improved its inhibitory activity by more than four orders of magnitude from the millimolar to the low nanomolar range, and we provided structural data for IDO1 binding of MMG-0706 (11) and MMG-0752 (12, Figure 1) 28 . These compounds feature a 2-amino, 5-halogen di-substituted phenyl ring and display a LE of 0.80 kcal/ mol/HA for the bromo compound, to our knowledge the highest LE reported for IDO1 inhibitors to date. Based on molecular modelling and quantum chemical calculations, we were able to explain the improved activity of the 2-amino substituent versus the 2hydroxy substituent in this scaffold, due to simultaneous intramolecular and intermolecular interactions 28 .
Here, we describe our efforts to improve and extend compounds comprising the 1,2,3-triazole and 1,2,4-triazole haem- binding scaffolds, the best new compound (144) demonstrating both enzymatic and cellular IC 50 values of 34 nM. Although this compound and our previously described compounds such as MMG-0358 (9), MMG-0706 (11), or MMG-0752 (12) 28,58 are highly efficient both in an enzymatic and in a cellular environment, they are very sensitive to even the smallest changes in their chemical structure, and therefore lack available sites to modulate their ADME and PK/PD properties. Analysing the active site structure of IDO1 and the chemical structures of other known IDO1 inhibitors, it would be natural to extend the compounds from pocket A in the haem distal site to pocket B towards the entrance of the active site ( Figure 1D). However, azole ligands are not optimally suited to be extended to pocket B due to their preferred orientation, and this extension often leads to a dramatic decrease in activity for many compounds 27 . Here, we resolve one new X-ray structure of a previously reported triazole extending to pocket B (13, MMG-0472), which validates our docking predictions. Based on this new structural data and the measured activities of almost 100 new compounds, we give recommendations for the development of future IDO1 inhibitors.

Results and discussion
Structural data MMG-0472-bound structure MMG-0472 (13, Figure 2) is a 4-phenyl-1,2,3-triazole featuring an extension on the phenyl ring, designed to be located in pocket B. We first described this compound in 2016, when we tested it in cellular assays for hIDO1 and for mIDO2 inhibition. MMG-0472 showed a high cytotoxicity (70% at 200 mM) and was not further pursued for this reason 62 . The mechanism behind this cellular toxicity remains to be clarified and could be due to solubility issues based on our experience with similar compounds. Here, we proceeded to test this compound in the enzymatic assay and found it to be one of the most potent azoles with B pocket extension with an enzymatic IC 50 value of 14 mM and a LE of 0.33 kcal/ mol/HA. We also investigated compound 13 by X-ray crystallography and obtained diffracting crystals of its complex with IDO1 (PDB ID 7zv3). The 2F o -F c map of the IDO1 bound ligand clearly shows its electron density in both pockets A and B (Figure 2A). The ligand is non-planar, with the central bond between the triazole ring and the chloro-phenyl displaying a dihedral angle of 39 . The His346-iron-ligand bond angle is 170 ( Figure 2B), deviating by 10 from its optimal value of 180 as determined by densityfunctional theory calculations on a haem model system ( Figure  2C). This deviation probably reflects some strain in the complex. The resolved ligand structure is very close to our docking prediction ( Figure 2D) with a root-mean-square distance (RMSD) of 0.3 Å.
As it can be appreciated from the top view and superimposition with the X-ray structures of triazoles 9, 11 and 12 ( Figure 2D,E), the phenyl ring in compound 13 is rotated away from Ser167 and towards pocket B by about 20 with respect to the phenyl rings of  the other compounds. Therefore, an additional 2-hydroxy substituent on the phenyl ring would not be favourable in these types of compounds, because they would produce a clash between the Bpocket extension and residues 261-265. The structural data for compound 13 thus confirms our previous analysis showing that azole ligands are not optimally suited to extend into pocket B 27 .

Enzymatic activities
Enzymatic IC 50 values were measured with the ascorbate/methylene blue reduction system 85 in presence of a non-ionic detergent to reduce compound aggregation as described before 28 . Kynurenine and L-Trp concentrations were determined by HPLC through UV detection. Dose-response curves and Hill slopes can be found in the Supplementary Information ( Figure S2). Generally, IC 50 values are slightly lower here than in our first work on 1,2,3triazoles 58 , as we shortened the incubation time to stop the reaction in its linear phase. If compound solubility allowed, compounds were tested at concentrations up to 1 mM in presence of 5% of DMSO co-solvent.

4-Aryl-1,2,3-Triazoles with monosubstituted phenyl groups
We previously showed that 1,2,3-triazoles with para-substituted phenyl rings consistently had lower inhibitory activities on IDO1 than unsubstituted compounds, their IC 50 values increasing from 10 mM (H) 28 to 190 mM (F), 530 mM (Cl), 1 mM (CH 3 ) to above 1 mM (CF 3 ) 58 for non-polar substituents. Here, we synthesised and tested one compound with the polar p-OH substituent (20), which was inactive (Table 1). This result shows that, in agreement with what has been found for imidazoles 41 , also polar substituents in this position are unfavourable. This is in agreement with docking predictions, showing little space and hydrophobic groups surrounding para substituents ( Figure 3A).
On the other hand, we have previously found that non-polar substitutions in meta position increased the activity and were most favourable for chloride (0.35 mM) 28 , while hydroxy (310 mM) and amino (>1 mM) substituents decreased activity 58 . Similar effects were found for substituents in this position in imidazoles 24,41 and hydroxyamidines 30 . Here, we tested more aliphatic substitutions of different sizes and found the ethyl substituent (22, 13 mM, Figure 3B) 58 to show the best activity, better than methyl (21, 22mM) 58 , n-butyl (24, 34mM), and i-propyl (23, 280 mM, Table 1). For larger substituents, there is not enough space in this position, as suggested by the structural analysis.
Earlier investigations of ortho substitutions in the 4-phenyl-1,2,3-triazoles showed that only the hydroxy substituent substantially increased the activity (29, 2.3 mM), attributed to the formation of a hydrogen bond with Ser167 in the back of the binding site 28 . Especially larger substituents, designed for targeting pocket 88 >100
B, showed substantially decreased inhibitory activities besides lowering the solubility of the compounds 58,62 . Here, we show that usage of an ether (25, 26, 27, Figure 3C) or an amino linker (28) to a large substituent in this position does not yield active compounds either (Table 1). This is also true when introducing a hydroxy group (27), expected to increase solubility and allowing  to test the inhibitor at higher concentrations. However, it should be noted that docking predicts that this hydroxy group cannot form favourable interactions within pocket B ( Figure 3D).

5-Substituted 4-Aryl-1,2,3-Triazoles
Theoretically, access to the B-pocket could also be achieved through a substitution either of the N3 or the C5 atoms of the 1,2,3-triazole ring ( Figure 1D). We explored the possibility of nitrogen substitutions before, without obtaining any active compound 58 . This finding supports the importance of an ionisable NH group in the triazole and the hypothesis that deprotonation of the 1,2,3-triazole is crucial for IDO1 inhibition. Here, we further explored substitution of the C5 atom (Table 2), although this enforces iron binding through the N2 atom ( Figure 1D), which has been calculated to be less favourable than binding through the N1 atom 28,58 . We previously described one compound of this type with a 5-methyl substituent (61, NI) 58 . Here, we show that an electron-withdrawing 5-nitrile substitution yielded a slightly active compound (62, 630 mM), while combination of the 5-methyl substituent with the 5-Cl substituent on the phenyl ring yielded an   50 value of 800 mM. The lower activities might be due to higher purities of our compounds and are in line with weak activities measured for other compounds of this type ( Table 2). Even combination with the highly activating 2-OH,5-Cl substitutions on the phenyl ring (67, 68) yielded at best a compound with an IC 50 value in the high micromolar range. These compounds cannot meaningfully be docked into the IDO1 active site, the best poses displaying a triazole-haem angle deviating significantly from the optimal angle of 90 (Supporting Information, Figure S1).

4-Aryl-Tether-1,2,3-Triazoles and annulated derivatives
The 1,2,3-triazole published by Alexandre and co-workers (10, Figure 1), which features an amino linker between the triazole and the 4-chlorophenyl moiety, shows a peculiar behaviour 60,70,86 . X-ray data demonstrates haem-iron binding and occupancy of pocket A ( Figure 4A), but a slow shift of the haem Soret peak in UV-absorption spectra under reductive conditions coupled with haem unbinding and degradation as well as the largely superior activity in cellular assays as compared to enzymatic assays is very distinct from the behaviour of the 4-aryl-1,2,3-triazoles 22,60 .
Building on these results, we synthesised and tested 1,2,3-triazoles linked to aryl moieties by a one-center tether (N, CH 2 , O, S, SO 2 , CHOH) or two-atom tether (CONH, Table 3). In our hands, compound 10 displayed an IC 50 value of 56 mM (11.3 mM in the original publication). 4-(4-Bromophenylamino)-1,2,3-triazole (78) showed an inhibitory activity of 52 mM, whereas the 4-phenylamino analogue (79) was not active. These 3 latter compounds dock well into the IDO1 active site, although the phenyl ring adopts a slightly rotated conformation with respect to the X-ray structure ( Figure 4B). Compound 80 with an additional phenyl substituent on the C5 of the triazole ring rather docks with the amino linker pointing towards the haem propionate and pocket B but shows no activity in the enzymatic assay (NI, Table 3, Figure  4C), similar to other C5-substituted 1,2,3-triazoles ( Table 2).
Replacement of the amino linker by a methylene group (81, NI), by an ether group (82, NI), by a sulphide group (83, 590 mM, Figure 4D), by a sulfoxide moiety (84, NI), by a hydroxymethylene group (85,NI) or by a carboxamide group (86, NI, Figure 4E) generated inactive compounds except for 83 with the sulphur tether (590 mM). Compound 87 with a MeON¼C linker was synthesised and tested as another possible access point to pocket B but was inactive. The annulated 1,2,3-triazoles 88 and 89 were also inactive. More interesting results were obtained with 5-aroyl-1,2,3triazoles (keto linker), preserving the conjugation and the planarity between the aromatic rings (Table 4). Docking results for compound 99 (430 mM) suggest that this compound can form a hydrogen bond with Ser167 ( Figure 4F), while the keto group simultaneously increases the acidity of the triazole ring. Starting from this 5-benzoyl-1,2,3-triazole scaffold, we explored different single and double substitutions of the phenyl ring, heterocyclic replacements of the phenyl ring, and 4-substitutions of the 1,2,3-triazole ring. Chloro substitutions in 2-position (100, NI), 3-position (101, 450 mM) and 4-position (102, 31 mM) of the phenyl ring showed that it was favourable only in the latter, in agreement with the analogous amino-1,2,3-triazole and the docking predictions ( Figure 4G   substituted compounds and provided the most active compounds of this type, which remained, however, only in the low micromolar range. In the docked poses, the hydroxy groups of these compounds form a hydrogen bond to Gly262 ( Figure 4H). Replacement of the phenyl ring by 5 or 6-membered aromatic heterocycles was mostly beneficial with 5-membered rings (115, 16mM, Figure  compounds, in agreement with the docking predictions, which do not find low-energy poses for these compounds inside the active site. In summary, we were able to develop original 5-aroyl-1,2,3-triazoles with enzymatic activities in the low micromolar range. However, they did not provide access to pocket B. 3-Aryl-1,2,4-Triazoles Since we did not find a 1,2,3-triazole providing a convenient access to pocket B while preserving efficiency and potency, we turned in the following to the 1,2,4-triazoles 28 . For this scaffold, we found previously that 3-(2-aminophenyl)-1,2,4-triazole (139) displayed an interesting inhibitory activity of 2 mM (Table 5). This was better than with the 3-(2-hydroxyphenyl) derivative (138, 31 mM), preferred by the 1,2,3-triazoles. Based on X-ray crystallography and molecular modelling studies, we attributed these results to the simultaneous inter-and intra-molecular hydrogen bonds of the 2-amino substituent in the 1,2,4-triazole, leading to a more favourable planar conformation ( Figure 5A). Most important, combination with the 5-chloro substituent (11, 0.035 mM) or the 5-bromo substituent (12, 0.020 mM) yielded highly active compounds with excellent LE of 0.78 and 0.80 kcal/mol/HA, respectively 28 .
Here, we started by synthesising and testing the para-fluorophenyl derivative (134), which was was found inactive and deterred us from preparing more para-substituted phenyl derivatives. Based on the knowledge collected for 1,2,3-triazoles, we tested halogen substitutions in meta position. We found that the bromo analogue (135, 16 mM) was more active than the chloro (136, 29 mM) and the fluoro (137, 160 mM) substituted compounds. This was expected based on the size of the hydrophobic subpocket in this region and in agreement with the activities found for 1,2,3-triazoles. A 2,6-diamino substituted compound (142, 31 mM, Figure 5C) showed decreased activity with respect to the singly substituted 2-amino compound (139, 2.0 mM). The 2-NH 2 ,5-F compound was consistently less active (143, 0.15 mM) than its other halogenated counterparts (compounds 11, 12).
We synthesised and tested two compounds with 2-amino,3halo disubstituted phenyl rings. As expected from our structural analysis, 140 (0.81 mM) and 141 (29 mM, Figure 5D) are much less active than their 2,5-disubstituted counterparts (Table 5). These compounds cannot offer any synergic effects from interactions of the amino group with Ser167 and the triazole ring on the one hand and the interactions of the halogen with the hydrophobic subpocket on the other hand. Methyl substitution of the amino group of compound 11 led to a lower inhibitory activity (145, 8.6 mM). This is in agreement with the structural data and the finding that both hydrogen atoms of the amino group serve as hydrogen bond donors 28 . We also synthesised and tested triple-substituted 3-phenyl-1,2,4-triazoles all having a 2-amino substitution of the phenyl ring. Adding a 4-F substitution to compound 12 only slightly perturbs its activity (144, 0.034 mM). However, adding a 6-methyl substitution to either compound 12 (155, 63 mM, Figure 5E) or 11 (156, 90 mM) reduced the inhibitory activities by more than 3 orders of magnitude. Adding a 6-amino group as in compound 157 (1 mM) also strongly reduced the inhibitory activity as compared to compound 11 (0.035 mM). This mirrors the results obtained with 2aminophenyl 139 and 2,6-diaminophenyl derivative 142 and leaves little chance to develop analogues of this type extending to pocket B.
Finally, we investigated amide extensions of the ortho-amino group with the hope that the latter could reach pocket B, although structural and functional data suggested this amino group to increase activity by being located in the back of the active site and making two simultaneous hydrogen bonds ( Figure  5A). We generally found that amide extensions reduced the compound solubility. With 3-(2-benzamidophenyl)-1,2,4-triazole (152), we lost the inhibitory activity completely ( Figure 5F). Curiously, introduction of a 5-Cl substituent restored some of the inhibitory activity (153, 9.4 mM, Table 5) albeit at low solubility. Interestingly, the cyclopropanecarboxamide (149, 0.49 mM) and the propenamide (147, 1.2 mM) showed the best inhibitory activities of this series. However, the related cyclobutanecarboxamide (150, NI) and cyclopentanecarboxamide (151, NI) were inactive as inhibitors and showed strongly reduced solubilities. In the aliphatic series, the butanamide (148, 24 mM) was better than the acetamide (146, 425 mM). Aromatic extensions such as the benzamide (153, 9.4 mM) led to lower solubilities. However, the latter could be improved by introducing polar groups. Unfortunately, the inhibitory activities of resulting compounds were only in the high micromolar range (data not shown). Summarising, the observed inhibitory activities of the 1,2,4-triazoles bearing 2-amido substituents cannnot be rationalised based on structural data. Investigations are rendered complicated by the low solubilities of these compounds.

Cellular activities
We tested the cellular hIDO1 inhibitory activity and toxicity of 30 active compounds at a single concentration ( Figure 6 and Table  8). As some of the aniline derivatives reacted with Ehrlich's reagent (p-dimethylaminobenzaldehyde, p-DMAB) used to quantify kynurenine, the kynurenine content of these samples was determined by HPLC. To be able to detect also weak inhibition, compounds were tested at the high concentration of 200 mM ( Figure  6A and B, filled bars), except for less soluble compounds (52, 148, 149 and 153), which were tested at a concentration of 50 mM ( Figure 6A and B, empty bars). Many compounds showed a good cellular inhibition ( Figure 6A) and a low toxicity ( Figure 6B). It is noticeable that for the compounds tested here, the 1,2,4-triazoles (orange) generally showed a better cellular inhibition than the 1,2,3-triazoles (black) but also a detectable albeit low toxicity. However, it should be kept in mind that previously tested 1,2,3-triazoles also showed a very good cellular inhibition 58 , and that the toxicity of the 1,2,4-triazoles occurs at a concentration of 200 mM, far above their cellular IC 50 value.
Previously determined cellular data for compounds mentioned in this work is given in the Supporting Information, Table S2. Both previously and in the present work, cellular inhibition was observed to be closely related to enzymatic activity for both 1,2,3-triazoles and 1,2,4-triazoles, showing a sigmoidal dependence ( Figure 6C). Three outliers show a lower cellular activity than expected, namely two double-substituted 5-aroyl-1,2,3-triazoles (112,113) and the fluorinated 1,2,4-triazole 143. On the other hand, three compounds show a higher cellular activity, namely compound 78 of the Vertex scaffold, for which this behaviour has already been documented 60 , the amide 1,2,4-triazole 146, which might be hydrolized to the highly active 11 inside the cell, and the 1,2,3-triazole 42 for unknown reasons.
Here, we determined cellular IC 50 values for a selection of three 1,2,4-triazoles ( Figure 6D), and found two of them to display nanomolar activities also in a cellular context. As in the enzymatic assay, the most potent compound is 144 (cellular IC 50 value of 0.034 mM), followed by 143 (0.25 mM) and 140 (1.2 mM).
We previously found a good correlation between enzymatic and cellular IC 50 values for different azole compounds 28,58 . Here, we show this correlation ( Figure 6E) for all 1,2,3-triazole (black) and 1,2,4-triazole (orange) compounds mentioned in this manuscript. The newly determined cellular IC 50 values of the 1,2,4- triazoles closely follow this correlation. The only outlier is the original Vertex compound 10 60 .

Conclusions
In summary, here we described almost 100 new compounds of the 1,2,3-triazole and the 1,2,4-triazole haem-binding series and tested them for their inhibitory activity on IDO1. They provide highly efficient scaffolds for inhibitors binding to pocket A, which are also very potent in a cellular environment and display low cytotoxicities. The best compound (144) displays both enzymatic and cellular IC 50 values of 34 nM and is therefore more potent and efficient in vitro than other frequently used IDO1 inhibitors.
We did not measure the activities of these compounds on IDO2 and on TDO to determine their selectivity. However, in our earlier works we found that triazoles such as MMG-0358 with high activities on IDO1 have undetectable activities on TDO (>100 lM) 58 . We also found that MMG-0358 has a more than1000-fold selectivity for IDO1 over IDO2, even though triazoles extending from pocket A to pocket B showed better activity on mouse IDO2 than on human IDO1 in a cellular environment 62 . Based on these observations, it is reasonable to assume that the potent compounds reported here, which are binding only to pocket A, are likely to be highly selective for IDO1 over TDO and IDO2.
Unfortunately, extending these highly efficient compounds into pocket B by one of the attachment points described in Figure 1(D) proved very challenging. We were able to resolve the X-ray structure of the complex of one such compound extending into pocket B (MMG-0472, PDB ID 7zv3). The experimental structure confirms our docking predictions of the binding mode of this compound. However, docking predictions in general are challenging due to the interactions with the haem cofactor in the active site, which is difficult to parameterise classically. For future design of type ii or type iii IDO1 inhibitors, we recommend tackling the issue of addressing pockets A and B simultaneously early on in hit-to-lead optimisation to provide more flexibility for rational compound modifications.

Experimental section
Docking Docking was performed with our in-house docking code AttractingCavities (AC) 87 , which relies on the physical scoring function of the CHARMM27 force field 88,89 , while solvation effects are taken into account by the Fast Analytical Treatment of Solvation (FACTS) model 90 , which has been shown to allow for accurate docking results 91 . Ligand force-field parameters were derived with the SwissParam tool 92 . Standard parameters were used, i.e. a cubic search space of 20 Å 3 around the IDO1 active site, a rotational angle of 90 for initial ligand sampling, and a N Thr value of 70 for determination of the attractive grid points. For all compounds with an acidic proton, both the neutral and the deprotonated species were docked, and different tautomers were considered. A Morse-like metal binding potential (MMBP) was used to describe the interactions between the haem iron of IDO1 and ligand atoms that display a free electron pair for iron binding 93 . The protein was kept fixed during the docking. We used seven different IDO1 X-ray structures as targets, chosen for their quality and diversity, namely PDB ID 2d0t 40 co-crystallized with a small azole ligand in pocket A, 5whr 25 with a resolved JK-loop C in closed conformation, 6e41 94 with an analogue of epacadostat in pockets A and B, 6kof 51 with a large azole ligand in pockets A and B, 6o3i 24 with the clinical compound navoximod, 6pu7 32 with a hydroxyamidine ligand of peculiar shape, and 7ah4 28 with two small azole ligands in pockets A and D. All ligands were removed before docking.

Density functional theory calculations
Quantum chemical geometry optimizations and charge calculations were carried out in the density functional theory (DFT) framework with the PBE0 hybrid functional 95 using the Gaussian16 code 96 . Geometry optimizations were carried out with standard settings and the TZVP basis set 97 . Solvation effects were taken into account by the polarisable continuum model 98 as implemented in Gaussian16. The histidine-bound haem complex of IDO1 was modelled by an iron-porphin-imidazole system. For the 6-fold coordinated systems, a low-spin complex was assumed, as it has been found experimentally 39 .

General remarks
All reactions were carried out under nitrogen atmosphere unless otherwise stated. Glassware was oven-dried (120 C), evacuated and purged with nitrogen. Any common reagents, catalysts and solvents that were obtained from commercial suppliers were used without any further purification. For extraction and chromatography, all solvents were distilled prior to use. Thin layer chromatography (TLC) for reaction monitoring was performed on silica gel plates (Merck 60 F254) with detection by UV light (254 nm). Flash chromatography (FC) was conducted using silica gel 60 Å, 230-400 mesh (Merck 9385). Mass spectra were recorded on a Nermag R10-10C instrument in chemical ionisation mode. Electrospray mass analyses were recorded on a Finnigan MAT SSQ 710 C spectrometer in positive ionisation mode. 1 H and 13 C NMR spectra were recorded with a Bruker-DPX-400 or Bruker-ARX-400 spectrometer at 400 MHz and 100.6 MHz, respectively. Data for 1 H NMR spectra are reported as follows: chemical shift, multiplicity, apparent coupling constant, and integration. Data for 13 C NMR spectra reported in terms of chemical shift. Chemical shifts are given in parts per million, relative to an internal standard such as residual solvent signals. Coupling constants are given in Hertz. Spectra were analysed with MestreNova. High resolution mass spectra were recorded via ESI-TOF-HRMS or MALDI-TOF-HRMS.
The purity of all final compounds was confirmed to exceed 95% by 1 H NMR showing 13 C-H satellite signals. Additionally, analytical HPLC purity analysis was carried out for compounds 34,113,143 and 149 with UV detection at 220 nm, using a PFP propyl column (RESTEK Allure HPLC column, particle size 5 mm, pore size 60 Å, dimensions 150 Â 4.6 mm) with a linear gradient of solvent B (acetonitrile, 0.1% TFA) over solvent A (H 2 O, 0.1% TFA) from 0 to 100% in 30 min at a flow rate of 1 mL/min. Typically, 25 mL solution (0.5 mg/ mL in 50% ACN/H 2 O) was injected for each compound.

General procedure (I) for the preparation of arylethynes
To a stirred solution of commercially available or synthetically prepared iodo derivatives (17) (Scheme 1 and 2) (1 eq) mixed with Et 3 N (4 eq) in dioxane (4 mL), trimethylsilylacetylene (1.3 eq), PdCl 2 (PPh 3 ) 2 (0.01 eq), and CuI (0.02 eq) were added. The reaction mixture was stirred at 45 C for 5 h under nitrogen atmosphere. After cooling to rt, the reaction mixture was diluted with Et 2 O (5 mL) and washed with brine (5 mL). The organic layer was dried (Na 2 SO 4 ), filtered, and concentrated under reduced pressure. KF (3.6 eq) was added to the residue and the mixture was dissolved in MeOH (5 mL) and stirred for 3 h at rt before concentration under reduced pressure. After addition of CH 2 Cl 2 (5 mL) and water (3 mL), the organic layer was collected, dried over MgSO 4 , and filtered through a short silica plug to afford the corresponding arylethynes 18 (Scheme 1). Overall yields 65-93%.

General procedure (II) for the preparation of 4-Aryl-1,2,3-Triazoles:
To a stirred solution of commercially available or synthetically prepared arylethynes (18) (1 equiv) and CuI (0.05 equiv) in DMF/ MeOH solution (2 mL, 9:1) under an argon atmosphere, TMSN 3 (1.5 equiv) was added. The resulting solution was stirred at 100 C for 10-12 h. After consumption of the ethynyl substrate, the mixture was cooled to rt, the precipitate was filtered off, and the remaining solution was concentrated under reduced pressure. The crude residue was purified by FC (SiO 2 , EtOAc/petroleum ether) to obtain the desired 4-aryl-1,2,3-triazole.
General procedure (III) for the demethylation of aryl methyl ethers: The methyl ether (1 eq) was dissolved in 48% HBr in water (4 mL), and the orange solution was heated to 100 C for 14 h under nitrogen atmosphere. The mixture was cooled to rt, diluted with water, and neutralised by the addition of a saturated aq. soln. of NaHCO 3 until the evolution of CO 2 ceased. The organic layer was extracted with EtOAc (2 Â 5 mL), dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure to afford a residue that was purified by FC (SiO 2 , EtOAc/petroleum ether) to give the desired phenol.  13   Water (6 mL) was added, and the aqueous layer was extracted with CH 2 Cl 2 to remove the oil and to give a transparent light colour solution. Finally, the aqueous solution was acidified with 10% HCl resulting in a light yellow precipitate which was washed with iced water to yield the crude product. 3-Chlorobenzaldehyde (280 mg, 2.0 mmol), nitromethane (180 mg, 2.4 mmol) and ammonium acetate (93 mg, 1.2 mmol) were added to 2 mL of glacial acetic acid. The resulting solution was heated under reflux for 2 h before pouring the reaction mixture into ice water. The yellow solid thus formed was collected by filtration to give the crude product 1-chloro-3-(2-nitroprop-1-en-1-yl)benzene (56a) 66,110 . The crude product (197 mg, 1.0 mmol) and NaN 3 (98 mg, 1.5 mmol) were stirred in DMF (3 mL), and p-TsOH (87 mg, 0.5 mmol) was added to the mixture at rt. The mixture was stirred at 60 C under air for 1 h. After completion of the reaction, the mixture was cooled to rt, quenched with H 2 O (5 mL) and extracted with EtOAc (3 Â 10 mL). The organic layers were collected, dried over anh. Na 2 SO 4 , filtered, and concentrated under reduced pressure. The residue was purified by FC (silica gel, EtOAc/hexane) to afford 63 (135 mg, 70%) as white solid, m.p. 161-163 C. 1  . The residue (58) (440 mg, 1.4 mmol) was dissolved in DMF (5 mL), Cs 2 CO 3 (764 mg, 2.2 mmol) was added, and the mixture was heated to 100 C, monitoring the progress of the reaction by TLC. The reaction mixture was cooled to rt and diluted with cold water. After extraction with EtOAc, the organic layers were collected, dried (anh. Na 2 SO 4 ), filtered, and concentrated under reduced pressure. The residue was purified by FC (silica gel, EtOAc/hexane) to give 64 (285 mg, 70%) as white solid. 1  After completion of the reaction, the mixture was cooled to rt and quenched with water. After extraction with EtOAc, the organic layer was collected, washed with brine, dried (anh. Na 2 SO 4 ), filtered, and concentrated under reduced pressure. Purification by FC (silica gel, EtOAc/hexane) produced 65 (150 mg, 60%) as white solid. 1 69 : To a stirred solution of 4-chlorobenzaldehyde (55c) (280 mg, 2.0 mmol), 2-cyanoacetamide (60) (169 mg, 2.0 mmol), and Et 3 NÁHCl (566 mg, 5.0 mmol) in DMF (3 mL), NaN 3 (390 mg, 6.0 mmol) was added. The mixture was stirred at 70 C for 10 h before cooling to rt and adding water (20 mL) and 10% HCl solution (1 mL). After extraction with EtOAc (2 Â 30 mL), the organic layer was collected, washed with water (3 Â 50 mL) and brine solution (1 Â 50 mL), dried (anh. Na 2 SO 4 ), filtered, and concentrated under reduced pressure. The residue was subject to FC (SiO 2 , CH 2 Cl 2 /MeOH) to obtain 66 (288 mg, 65% yield) as white solid, 1

Synthesis
of 4-Aryl-Tether-1,2,3-Triazoles and annulated derivatives N-(4-Bromophenyl)-1,2,3-triazol-4-amine (78) 71 : 4-Bromo aniline (335 mg, 2.62 mmol) was dissolved in hydrochloric acid (6.03 mL of 2 M, 12.06 mmol), diluted with water (8 mL) and cooled to 0 C. Sodium nitrite (181 mg, 2.62 mmol) was added and the mixture was stirred at 0 C for 20 min before slowly adding a solution of 2aminoacetonitrile monohydrochloride (70) (243 mg, 2.62 mmol) in water (3 mL). The mixture was stirred for 10 min at 0 C before adding sodium acetate (3.02 g, 37.0 mmol) and allowing the mixture to warm to rt, where it was stirred for 1 h. The precipitate was collected by filtration and washed with water to afford 2-(3-(4-chlorophenyl)triaz-2-en-1yl)acetonitrile. The residue (238 mg, 1.0 mmol) was dissolved in EtOH (7 mL) and heated under reflux for 4 h. The mixture was allowed to cool to rt and concentrated under reduced pressure. The crude compound was triturated with CH 2 Cl 2 to produce a cream solid which was purified by FC (40% EtOAc in petroleum ether) to afford 78 (167 mg, 70%) as a white solid. 1 111 : To a stirred solution of compound 83 (60 mg 0.34 mmol) in methanol (2 mL), H 2 O 2 (0.1 mL, 2.7 mmol) and a catalytic amount of ammonium molybdate (7 mg, 0.04 mmol) were added and allowed to stir at rt for 14 h, monitoring the progress of the reaction by TLC. After completion of the reaction, the solvent was removed under reduced pressure. The obtained solid products were dissolved in CH 2 Cl 2 (2 mL) and water (2 mL), and the aqueous phase was extracted with CH 2 Cl 2 (2 Â 4 mL). The combined organic layers were washed with brine, dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The residue was purified by FC to give 84 (57 mg, 80%) as a white solid. 1 3.0 mmol) and potassium hydroxide (168 mg, 3.0 mmol) were dissolved in diethyleneglycol (3 mL) under nitrogen atmosphere. The reactions mixture was heated to 170-190 C and maintained at this temperature for 6 h. Upon completion of the reaction, the mixture was cooled to rt, and water (5 mL) and EtOAc (10 mL) were added. EtOAc (10 mL) was added, the phases were separated, and the aqueous phase was further extracted with EtOAc (3 Â 10 mL). The combined organic phases were washed with brine, dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The residue was purified by FC to yield 89 (63 mg, 40%) as a white solid. 1 115 : To a solution of TMS-acetylene (75) (655 mg, 3.0 mmol) in 10 mL THF at À78 C, n-BuLi (2.5 M in hexane, 1.1 mL, 3.0 mmol) was added dropwise and stirred for 1.5 h. A solution of diphenyl disulphide (74) (324 mg, 3.3 mmol) in 2 mL THF was added dropwise, and the mixture was warmed to rt over 2 h before adding H 2 O (5 mL). After extraction with Et 2 O (3 Â 10 mL), the organic layer was collected, washed with brine, dried over Na 2 SO 4 ), filtered, and concentrated under reduced pressure to yield the TMS-protected thioethyne. For deprotection, the thioethyne (412 mg 2.0 mmol) was stirred for 3 h at rt with KF (441 mg, 7.6 mmol) in MeOH (10 mL). After concentration under reduced pressure, CH 2 Cl 2 (5 mL) and water (3 mL) were added. The organic layer was collected, dried (MgSO 4 ) and filtered through a short silica plug to afford 77b (192 mg, 85%) as a brown oil. 1  N-Phenylpropiolamide (77d) 116 : Propiolic acid (350 mg, 5.0 mmol) and DCC (1.03 g, 5.0 mmol) were combined in 15 mL of CH 2 Cl 2 . The solution was stirred for 10 min before adding aniline (76) (466 mg, 5.0 mmol) and DMAP (7.5 mg, 0.06 mmol) in dry CH 2 Cl 2 at 0 C under nitrogen atmosphere. After complete addition, the mixture was stirred at rt for 18h. The reaction mixture was filtered and washed with 1 N HCl (5 mL) and a saturated solution of sodium chloride (2 Â 50 mL). The organic phase was separated, dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The residue was purified by FC (silica gel, EtOAc/hexane) to provide 77d (652 mg, 90%) as a colourless oil. General procedure (IV) for the synthesis of (aryl)(1,2,3-Triazolyl)methanol Ethynylmagnesium bromide solution (1.2 eq, 0.5 M solution in THF) was added dropwise to a stirred solution of aldehydes (90) (1 eq) in anhydrous THF (6 mL) at 0 C. The mixture was kept stirring at 0 C for 20 min, then warmed to rt and stirred for an additional 2 h. The reaction mixture was quenched with a saturated NH 4 Cl solution (6 mL) and extracted with EtOAc (3 Â 8 mL). The organic layers were combined, dried over anh. Na 2 SO 4 , filtered, and concentrated under reduced pressure to give the intermediate 1-aryl propargyl alcohols (91), which were directly used in next step. Alcohols (91) (1 eq), TMSN 3 (1.5 eq), and CuI (0.05 eq) in DMF/MeOH solution (9:1) were stirred at 100 C for 10-12 h. After consumption of the ethynyl substrate, the mixture was cooled to rt, the precipitate was filtered off, and the solution was concentrated under reduced pressure. The crude residue was purified by FC (SiO 2 , EtOAc/petroleum ether) to afford compounds 92a-92u in 55-85% yields.

Protein expression and purification
Full-length human IDO1 gene was cloned into the pET-28a vector and expressed in E. coli Rosetta (DE3) cells. Protein was produced in LB medium supplemented with 0.5 mM dALA (5-aminolevulinic acid) and was induced by addition of 0.1 mM isopropylthio-b-galactoside overnight at 16 C. The culture was harvested and frozen at À80 C until further use. The frozen cells were lysed by sonication in 20 mM Tris pH 7, 400 mM NaCl, 1 mM MgCl 2 , 20 mM imidazole, 1 mM DTT, 0.5 mg/mL Lysozyme and Protease inhibitor. After centrifugation, the supernatant was loaded into a HisTrap HP column pre-equilibrated with buffer containing 20 mM Tris pH 7, 500 mM NaCl and 20 mM imidazole, and eluted with the same buffer containing 500 mM imidazole. Fractions containing IDO1 were pooled, concentrated, and injected on a size exclusion chromatography column (HiLoad Superdex S75 16/60) to increase purity. hIDO1 fractions were concentrated to 20 mg/mL in 20 mM Tris pH 7 and 150 mM NaCl.
Protein crystallisation, data collection, structure determination and model refinement hIDO1 protein at 20 mg/mL was mixed with compound MMG-0472 at a final concentration of 5 mM, and co-crystallized by sitting drop vapour diffusion method. Crystals formed in a couple of days in 15% PEG 4000, 0.2 M lithium sulphate, and 0.1 M Tris pH 7.5. The crystals were cryoprotected with 25% glycerol. Diffraction data were collected at the Paul Scherrer Institute (SLS, Villigen) at PXII-X06DA beamline. Data were processed with the XDS Program Package 126 . Structures were solved by molecular replacement using Phaser-MR and chain A of PDB entry 2d0t 40 as the model. Manual model building and structure refinement were carried out in Phenix Suite 127 using coot software 128 and phenix-refine, respectively. After validation, the ligand bound IDO1 model was deposited in the PDB database under PDB code 7zv3. Data collection and refinement statistics are summarised in the Supplementary Information (Table S1). The structures were displayed with PyMOL (http://www.pymol.org/) and UCSF Chimera 129 . Authors will release the atomic coordinates and experimental data upon article publication.

Enzymatic assays
The enzymatic inhibition assays were performed as described by Takikawa et al. 130 with some modifications. Briefly, the reaction mixture (100 mL) contained potassium phosphate buffer (100 mM, pH 6.5) ascorbic acid (20 mM), catalase (400 units/mL), methylene blue (10 mM), purified recombinant IDO1 (2.5 ng/mL), L-Trp (100 mM), Triton X-100 (0.01%) and DMSO (5 mL). The inhibitors were serially diluted 3-fold for at least 8 different concentrations. After incubation at room temperature for 20 min, the reaction was stopped in its linear phase by addition of trichloroacetic acid solution (40 mL, 30% w/v), and the samples were incubated at 50 C for 30 min. After centrifugation, 80 ml of supernatant from each well were used for HPLC analysis. The mobile phase for HPLC analysis was composed of 50% (v/v) of methanol and 50% of sodium citrate buffer (40 mM, pH 2.4) containing 400 mM sodium dodecyl sulphate. An Agilent Zorbax Eclipse XDB C18 column (150 Â 4.6 mm) was used at 23 C with a flow rate of 1 mL/min and an injection volume of 20 mL. For detection of kynurenine, absorption at 365 nm was measured, while remaining L-Trp was detected at a wavelength of 280 nm. All assays were carried out in duplicates. The activity (Act) of each compound was calculated as Act ¼ RT log(IC 50 ) in analogy to the relation between the binding free energy and the K i value. The ligand efficiency (LE) was calculated as LE ¼ ÀAct/N HA , where N HA is the number of non-hydrogen atoms (heavy atoms) in the compound.

Cellular assays
The cDNA encoding hIDO1 (NCBI, NM 0021464.3) was purchased from Origene as pCMV6-Entry vector (RC206592). The full ORF of hIDO1 was cloned into the mammalian expression vector pcDNA6/myc-His from Invitrogen. Human embryonic kidney 293 T (HEK 293 T) cells were transiently transfected with the plasmid construct encoding hIDO1. A 75 cm 2 flask containing HEK 293 T cells at a confluency of 70% was transfected with jetPEI DNA transfection reagent (Polyplus-transfection), according to manufacturer's protocol. A total of 20 mg of pcDNA-IDO1 along with 5 mg of pcDNA-GFP plasmid were used for the transfection; GFP was used to evaluate transfection efficiency prior to the cellular assays. 16-18 h post-transfection the 293 T cells were detached with trypsin, centrifuged, and re-suspended in 25 mL of DMEM medium without phenol red, supplemented with penicillin and streptomycin, 10% foetal bovine serum and 1 mM pyruvate (Gibco). Subsequently, 100 mL of cells were distributed to each well of two 96-well round-bottom plates that had been pre-loaded with 100 mL DMEM and the small organic test molecule in DMSO (for a final DMSO concentration of 2%). DMEM medium contains 78 mM L-Trp and was supplemented with 500 mM of L-Trp. Cells transfected with hIDO1 were incubated for 7 h at 37 C in a CO 2 incubator. The reaction was stopped in its linear phase by adding trichloroacetic acid (TCA) to the medium at a final concentration of 4%. Plates were centrifuged for 15 min at 4,000 rpm. 100 mL of supernatant was added to 100 mL of 2% (w/v) DMAB in glacial acetic acid. After 10 min the absorbance was measured at a wavelength of 480 nm to detect kynurenine formation 85 . Alternatively, kynurenine content was determined by HPLC as done for the enzymatic assay.

Cell viability
Following DMSO (negative control) or inhibitor treatments, apoptotic cells were detected by 4 0 ,6-diamidino-2-phenylindole (DAPI) staining. Briefly, after 7 h of incubation, cells were washed with PBS, resuspended in PBS with 1 mg/mL DAPI and immediately analysed using a BD LSR II cytometer. A 405 nm laser with 450/50 nm bandpass filter was used to collect data.